JP6991572B2 - Barrier function measurement system for artificial 3D tissue, barrier function measurement method for artificial 3D tissue, and drug evaluation method using artificial 3D tissue - Google Patents

Description

The present invention relates to a barrier function measuring system for an artificial three-dimensional tissue, a barrier function measuring method for an artificial three-dimensional tissue, and a drug evaluation method using an artificial three-dimensional tissue.

In recent years, in the development of drugs, a model of the blood-brain barrier (BBB), which is a barrier function on the wall surface of a blood vessel, for example, a barrier function on the wall surface of a cerebral blood vessel targeting the central nervous system is required. Transendothelial electrical resistance (TEER), which is the electrical resistance between the lumen side and the outside of the lumen, is widely used as an index for evaluating the barrier function.

In Non-Patent Document 1, vascular endothelial cells are arranged on one side, and electrodes are arranged on both sides of a planar porous membrane in which nerve cells (astrocytes) are arranged on the other side, and electrical resistance between the electrodes is provided. The technology for measuring is disclosed.

Y. Wang, et al, Biotechnology and Bioengineering (2017)

In the technique described in Non-Patent Document 1, since vascular endothelial cells and nerve cells are arranged two-dimensionally, the flow of the medium flowing on the vascular endothelial cell side tends to be uneven. Therefore, the measured electrical resistance may become non-uniform in the flow direction of the medium, and as a result, the reliability of the obtained transendothelial electrical resistance and the evaluation of the barrier function may decrease.

The present invention has been made in view of the above problems, and is an artificial three-dimensional tissue barrier function measuring system capable of measuring transendothelial electrical resistance and evaluating the barrier function with high reliability, and a barrier function of an artificial three-dimensional tissue. It is an object of the present invention to provide a measurement method and a drug evaluation method using an artificial three-dimensional tissue.

According to the first aspect of the present invention, a medium is supplied to a tubular cavity having a perfusion channel extending in a predetermined direction, a tissue portion arranged outside the cavity, and the perfusion channel. A supply device, a flow path electrode arranged in the perfusion flow path, a tissue electrode arranged in the tissue portion, and a measurement device for measuring transendothelial electrical resistance via the flow path electrode and the tissue electrode. A barrier function measuring system for an artificial three-dimensional tissue is provided.

According to the second aspect of the present invention, an artificial three-dimensional tissue having a tubular cavity portion having a perfusion flow path extending in a predetermined direction and a tissue portion arranged outside the lumen portion is formed. It includes supplying a medium to the perfusion channel and measuring transendothelial electrical resistance via a channel electrode arranged in the perfusion channel and a tissue electrode arranged in the tissue portion. A method for measuring the barrier function of an artificial three-dimensional tissue is provided.

According to the third aspect of the present invention, the barrier function of the artificial three-dimensional tissue is measured by the measuring method of the second aspect of the present invention, the drug is brought into contact with the artificial three-dimensional tissue, and the drug is used. Provided is a method for evaluating a drug using an artificial three-dimensional tissue, which comprises measuring the response of the artificial three-dimensional tissue to a stimulus by contact with the artificial three-dimensional tissue.

According to the present invention, a barrier function measuring system for artificial three-dimensional tissues, a barrier function measuring method for artificial three-dimensional tissues, and a drug evaluation method using artificial three-dimensional tissues, which can obtain highly reliable transendothelial electrical resistance and barrier function. Can be provided.

It is a perspective sectional view schematically showing the artificial three-dimensional structure 1 which concerns on embodiment of this invention. It is an external perspective view of the perfusion device 40 in the pre-stage of forming (culturing) an artificial three-dimensional tissue. It is sectional drawing which cut the perfusion device 40 in the plane which includes the flow path forming member 43 in the length direction. It is sectional drawing of the perfusion device 40 after moving a flow path forming member 43. It is a front view which looked at the engaging part 42c in the axial direction. FIG. 5 is a cross-sectional view taken along the line AA in FIG. It is a figure which connected the flow path electrode 70 and the tissue electrode 80 to the measuring part 90. It is a schematic block diagram of an artificial three-dimensional tissue 1. It is a figure which shows the manufacturing procedure of the artificial three-dimensional structure 1. It is a figure which shows the manufacturing procedure of the artificial three-dimensional structure 1. It is a figure which shows the manufacturing procedure of the artificial three-dimensional structure 1. It is a figure which shows the manufacturing procedure of the artificial three-dimensional structure 1. It is a figure which shows the relationship between the culture time and transendothelium electrical resistance (TEER) in the artificial three-dimensional tissue 1.

Hereinafter, embodiments of the barrier function measuring system for artificial three-dimensional tissues, the barrier function measuring method for artificial three-dimensional tissues, and the drug evaluation method using artificial three-dimensional tissues of the present invention will be described with reference to FIGS. 1 to 13. explain.

It should be noted that the following embodiments show one aspect of the present invention, do not limit the present invention, and can be arbitrarily changed within the scope of the technical idea of the present invention. Further, in the following drawings, in order to make each configuration easy to understand, the scale and number of each structure are different from the actual structure.

(Barrier function measurement system)
First, the barrier function measuring system according to the present invention will be described with reference to FIG.
FIG. 1 is a schematic configuration diagram of a barrier function measuring system 100.
As shown in FIG. 1, the barrier function measuring system 100 includes a perfusion device (device) 40, a culture dish 50, a pump (supply device) 60, a medium reservoir 61, a filter 62, a flow path electrode 70, a tissue electrode 80, and a measuring unit. It has 90.

The perfusion device 40 is placed in the internal space of the culture dish 50. The medium reservoir 61 stores the medium M. The pump 60 supplies the medium M stored in the medium reservoir 61 to the perfusion device 40 via the pipe 63. The medium M discharged from the perfusion device 40 is supplied to the perfusion device 40 after the foreign matter is removed by the filter 62. As the pump 60, a peristaltic pump is used as an example.

(Perfusion device 40)
FIG. 2 is an external perspective view of the perfusion device 40 in the pre-stage of forming (culturing) an artificial three-dimensional tissue.
The perfusion device 40 includes a culture tank 41, a connector (support portion) 42, a flow path forming member 43, and a tissue electrode 80. The culture tank 41 includes a culture space 45 surrounded by a side wall 44 and an open upper portion, and a bottom plate 47 provided on the bottom wall 46. The side wall 44 is provided in a rectangular shape in a plan view. The bottom plate 47 can be attached to and removed from the culture tank 41. The bottom plate 47 can open and close the opening provided in the bottom wall 46 of the culture tank 41.

A plurality of pairs (6 pairs in FIG. 2) of the connectors 42 are mounted at positions where the through holes 42d are coaxial with each of the facing side walls 44. The three pairs of connectors 42 are arranged along the first direction, and the other three pairs of connectors 42 are arranged along the second direction which is horizontally orthogonal to the first direction. Of the connector 42, at least the region exposed to the culture space 45 is subjected to a liquefaction treatment for extracellular matrix component 11 (described later; see FIGS. 6 and 8). As the liquefaction treatment, for example, O ₂ plasma treatment can be adopted.

In the present embodiment, a case will be described in which the flow path forming member 43 and the flow path electrode 70 are inserted into one of two adjacent pairs of connectors 42 among the six pairs of connectors 42, and the tissue electrode 80 is inserted into the other. do. In the following, the tissue electrode 80 is inserted and supported by the connector (first support portion) 42A, and the flow path forming member 43 and the flow path electrode 70 are inserted and supported by the connector (second support portion) 42B. Explain as a thing.

FIG. 3 is a cross-sectional view of the perfusion device 40 cut at a surface including the flow path forming member 43 in the length direction.
As shown in FIG. 3, the connectors 42A and 42B penetrate the inside in the extending direction (the direction penetrating the side wall 44) of the mounting portion (cylinder portion) 42a, the connecting portion 42b, the engaging portion 42c, and the flow path forming member 43. It has a through hole 42d to be formed. The mounting portion 42a is formed in a shaft shape and is mounted so as to penetrate the side wall 44 of the culture tank 41. The connecting portion 42b is provided at one end of the mounting portion 42b. The connection portion 42b can be connected to the pipe 64 arranged outside the culture tank 41.

In the stage before forming (culturing) the artificial three-dimensional tissue, the flow path forming member 43 is movably inserted in the through hole (insertion hole) 42d of the connector 42B in the length direction and suspended in the culture space 45. .. As an example, the flow path forming member 43 is formed in a shaft shape having an outer diameter of 0.5 mm. The flow path forming member 43 is formed so as to have an end portion protruding from the connector 42B (connecting portion 42b). One end of the flow path electrode 70 is connected to one end of the flow path forming member 43.

FIG. 4 is a cross-sectional view of the perfusion device 40 after moving the flow path forming member 43.
As shown in FIG. 4, the flow path electrode 70 is suspended in the culture space 45 by moving the flow path forming member 43 to the other end side (left side in FIG. 4) in the length direction.

As shown in FIGS. 3 and 4, the pipe 64 has an insertion portion 65 having an insertion hole 65a through which the flow path electrode 70 connected to the measurement unit 90 is inserted, and the flow path electrode 70 is suspended in the culture space 45. When this is done, as shown in FIG. 1, it has a bifurcated structure branched into a medium supply unit 66 having a supply hole 66a to which the medium is supplied via the pipe 63.

In FIGS. 3 and 4, the pipe 64 is connected to only one of the opposing connectors 42 on which the flow path forming member 43 and the flow path electrode 70 are suspended, but in reality, the opposing connectors 42A, It is connected to both of 42B respectively.

The engaging portion 42c is provided at the other end of the mounting portion 42b. The engaging portion 42c is arranged in the culture space 45 of the culture tank 41 with a gap from the side wall 44. FIG. 5 is a front view of the engaging portion 42c as viewed in the axial direction. FIG. 6 is a cross-sectional view taken along the line AA in FIG. As shown in FIGS. 5 and 6, the engaging portion 42c is spaced coaxially with the outer peripheral surface of the mounting portion 42a from the second tubular portion 42e and the mounting portion 42a in the circumferential direction. It is provided with a plurality of arranged rib portions 42f. The rib portion 42f connects the outer peripheral surface of the mounting portion 42a and the inner peripheral surface of the second tubular portion 42e. Four rib portions 42f are provided at 90-degree intervals. The gap 42g surrounded by the mounting portion 42a, the second cylinder portion 42e, and the rib portion 42f penetrates the engaging portion 42c in the axial direction.

FIG. 7 is a diagram in which the flow path electrode 70 and the tissue electrode 80 are connected to the measuring unit 90.
As shown in FIG. 7, the flow path electrode 70 has a first flow path electrode 71 and a second flow path electrode 72. The first flow path electrode 71 and the second flow path electrode 72 form a first stranded wire 73 twisted to each other. The tissue electrode 80 has a first tissue electrode 81 and a second tissue electrode 82. The first structure electrode 81 and the second structure electrode 82 form a second stranded wire 83 twisted to each other. The maximum outer diameter of the first stranded wire 73 is formed to be smaller than the outer diameter of the flow path forming member 43.

As the first flow path electrode 71, the second flow path electrode 72, the first tissue electrode 81, and the second structure electrode 82, a metal wire rod made of Ag, AgCl, platinum, gold, carbon, or the like can be used. Of the first flow path electrode 71 and the second flow path electrode 72 constituting the first stranded wire 73, the outer peripheral surface of the second flow path electrode 72 is insulated and coated. A metal wire is exposed on the end face of the second flow path electrode 72. Of the first structure electrode 81 and the second structure electrode 82 constituting the second stranded wire 83, the outer peripheral surface of the second structure electrode 82 is insulated and coated. A metal wire is exposed on the end face of the second structure electrode 82. This makes it possible to suppress a short circuit between the first flow path electrode 71 and the second flow path electrode 72 and a short circuit between the first tissue electrode 81 and the second tissue electrode 82. The outer peripheral surfaces of the second flow path electrode 72 and the second tissue electrode 82 are, for example, insulated and coated with polyparaxylylene (hereinafter referred to as parylene).

(Artificial 3D tissue)
Next, the artificial three-dimensional structure will be described with reference to FIG. FIG. 8 is a schematic configuration diagram of the artificial three-dimensional organization 1.
As shown in FIG. 8, the artificial three-dimensional tissue 1 includes a tubular lumen portion 15 having a perfusion flow path 13 and a tissue portion 10 arranged outside the lumen portion 15. The tissue portion 10 in the present embodiment is formed of the extracellular matrix component 11.

The extracellular matrix component 11 is not particularly limited, and is, for example, collagen (type I, type II, type III, type V, type XI, etc.), mouse EHS tumor extract (type IV collagen, laminin, heparan sulfate proteoglycan, etc.). (Including), basement membrane components reconstituted (trade name: Matrigel), gelatin, agar, agarose, fibrin, glycosaminoglycan, hyaluronic acid, proteoglycan and the like can be exemplified.

The perfusion flow path 13 is a flow path through which the medium is perfused, and penetrates the inside of the tissue portion 10 and extends in the first direction. The luminal portion 15 is formed by using vasculature cells 14 on the surface of the tissue portion 10 facing the perfusion flow path 13. As the vasculature cell 14, for example, endothelial cells can be used.

Examples of the vascular lineage cells include vascular epithelial cells and vascular endothelial cells, and vascular endothelial cells are preferable. Examples of vascular endothelial cells include cells derived from mammals such as humans, mice, and rats, and human-derived vascular endothelial cells are preferable. Human-derived vascular endothelial cells include Human Umbilical Vein Endothelial Cells (HUVEC), Human Umbilical Artery Endothelial Cells (HUAEC), and Human Coronary Artery Endothelial Cells: HCAEC), Human Saphenous Vein Endothelial Cells (HSaVEC), Human Pulmonary Artery Endothelial Cells (HPAEC), Human Aortic Endothelial Cells (HAoEC), Human Skin Microvascular Human Dermal Microvascular Endothelial Cells (HDMEC), Human Dermal Blood Endothelial Cells (HDBEC), Human Dermal Lymphatic Endothelial Cells (HDLEC), Human Lung Microvascular Endothelial Cells (HDLEC) Human Pulmonary Microvascular Endothelial Cells (HPMEC), Human Cardiac Microvascular Endothelial Cells (HCMEC), Human Bladder Microvascular Endothelial Cells (HBdMEC), Human Uterine Microvascular Endothelial Cells: HUtMEC), human brain microvascular endothelial cells and the like.

(Artificial three-dimensional structure manufacturing method)
Next, a method for manufacturing the artificial three-dimensional structure 1 will be described with reference to FIGS. 9 to 12.
The method for producing an artificial three-dimensional tissue 1 according to the present invention is a flow in which a culture tank 41 having a culture space 45 surrounded by a side wall 44 and a flow suspended in a culture space 45 through the opposite side wall 44 in a predetermined direction. A perfusion device (artificial three-dimensional tissue perfusion device, device) 40 provided with a path forming member 43 and a tissue electrode 80 is prepared, and the extracellular matrix component 11 is cultured in the culture space 45 to form a flow path forming member 43. And forming the tissue portion 10 through which the tissue electrode 80 penetrates, removing the flow path forming member 43 from the tissue portion 10 to form a perfusion channel 13 penetrating the tissue portion 10, and flowing into the perfusion channel 13. It includes arranging the path electrode 70 and seeding vascular cells on the surface of the tissue portion 10 facing the perfusion channel 13 to form the lumen portion 15.

Hereinafter, a method for manufacturing an artificial three-dimensional structure will be described in detail.
In FIGS. 9 to 12, only the perfusion device 40 is shown as appropriate, and the culture dish 50, the pump 60, and the like are omitted.

(Preparation of perfusion device 40)
As a preparation for the perfusion device 40, as shown in FIGS. 2 and 9, connectors 42A and 42B are attached to the facing side walls 44 of the culture tank 41 so that the through holes 42d are coaxial. The flow path forming member 43 is inserted through the through hole 42d of the coaxial connector 42B, the tissue electrode 80 is inserted through the through hole 42d of the connector 42A, and the flow path forming member 43 and the tissue electrode 80 are suspended in the culture space 45. Let me.

In order to seal the gap between the attachment portions of the connectors 42A and 42B to the side wall 44 and the attachment portion of the bottom plate 47 to the bottom wall 46, the surface of the culture tank 41 facing the culture space 45 is coated with a sealing material. As the sealing material, a material that does not adversely affect the extracellular matrix component 11, for example, polyparaxylylene (hereinafter referred to as parylene) is formed by a film forming method such as thin film deposition. The film thickness of the sealing material is preferably 1/100 to 1/10 with respect to the gap between the mounting portions and the gap between the mounting portions. The film thickness of the sealing material is preferably 1/50 to 1/10, more preferably 1/50 to 1/20 with respect to the gap between the mounting portions and the gap between the mounting portions. In the present embodiment, a sealing material is formed with a film thickness (1/25) of 2 μm with respect to a gap of about 50 μm.

Of the connectors 42A and 42B, at least the region exposed to the culture space 45 is subjected to the liquefaction treatment. The liquefaction treatment may be carried out on the connectors 42A and 42B before being attached to the culture tank 41, or may be carried out on the connectors 42A and 42B mounted on the side wall 44.

(Formation of tissue portion 10)
When the preparation of the perfusion device 40 is completed, the tissue portion 10 is formed. For the formation of the tissue portion 10, as shown in FIG. 9, the solution of the extracellular matrix component 11 is poured into the culture space 45 of the culture tank 41. The extracellular matrix component 11 is poured in an amount that is high enough to immerse the flow path forming member 43 and the tissue electrode 80. In this embodiment, collagen is used as the extracellular matrix component 11, and a mixed solution of 10-fold concentration PBS and cell culture solution is used, and the mixing ratio is 9: 1: 5. As the cell culture medium, for example, EGM, which is a culture medium for vascular endothelial cells, is used. Further, the mixing ratio does not necessarily have to be the above ratio as long as the collagen solution is gelled.

When the extracellular matrix component 11 is poured into the culture tank 41, it is cultured (incubated) under predetermined conditions. As an example, the culture conditions were carried out at a temperature of 37 ° C. for 30 to 60 minutes.

The extracellular matrix component 11 gels by culturing. When the extracellular matrix component 11 gels and the culture is completed, the tissue portion 10 through which the flow path forming member 43 penetrates is formed. Further, in the tissue portion 10, as shown in FIG. 11 which is a partial plan sectional view, the tissue electrode 80 penetrates in parallel with the flow path forming member 43 at intervals.

When cells are contained in the extracellular matrix component 11, the extracellular matrix component 11 contracts by further continuing the culture. Since the extracellular matrix component 11 is engaged with the engaging portion 42c at the time of contraction, the contraction in the stacking direction is not constrained, but the contraction in the first direction and the second direction is constrained. More specifically, as shown in FIG. 6, the extracellular matrix component 11 is engaged with the second tubular portion 42e and the rib portion 42f of the engaging portion 42c from the side opposite to the center of the culture space 45. Therefore, the second tubular portion 42e and the rib portion 42f serve as barriers to suppress the contraction toward the center side. Further, when the extracellular matrix component 11 contracts, it is crimped to the outer peripheral surface of the second tubular portion 42e and the outer peripheral surface on the tip end side of the mounting portion 42a due to the contraction in the stacking direction. Therefore, the frictional force between the extracellular matrix component 11 and the engaging portion 42c increases, and the resistance to contraction in the first direction and the second direction increases. In particular, in the present embodiment, since the engaging portion 42c is subjected to the liquefaction treatment with respect to the extracellular matrix component 11, the extracellular matrix component 11 adheres to the engaging portion 42c with a larger adhesion force in the first direction. And the resistance to shrinkage in the second direction increases. Therefore, even if the culture is performed under the condition that the extracellular matrix component 11 contracts, such as when cells are contained, the extracellular matrix component 11 can be stably supported.

(Formation of perfusion channel 13 and lumen 15)
Next, the flow path forming member 43 supported by the connector 42B is removed. Specifically, the flow path forming member 43 is moved to the side opposite to the one end side (on the left side in FIG. 10) to which the flow path electrode 70 is connected. By moving the flow path forming member 43 to the other end side, a perfusion flow path 13 which is a cavity extending linearly with a diameter corresponding to the outer diameter of the flow path forming member 43 is formed in the tissue portion 10. At this time, since the tissue portion 10 is engaged with the engaging portions 42c at both ends, it is stably supported by the connector 42B without being detached from the connector 42B when the flow path forming member 43 is removed.

Further, as the flow path forming member 43 moves to the other end side, the flow path electrode 70 connected to one end side of the flow path forming member 43 is inserted into the perfusion flow path 13 as shown in FIG. To. Further, one end side of the flow path electrode 70 connected to the flow path forming member 43 is separated from the flow path forming member 43 and arranged in, for example, the through hole 42d of the connector 42B.

The second tissue electrode 82 in the tissue electrode 80 is insulated and coated, but since the end face is exposed, it is in contact with the tissue portion (collagen) 10. Further, although the second flow path electrode 72 in the flow path electrode 70 is insulated and coated, the end surface separated from the flow path forming member 43 is exposed and is arranged in the through hole 42d communicating with the perfusion flow path 13. ..

Next, as shown in FIG. 12, the above-mentioned HUVEC as vasculature cells 14 is injected (seed) into the surface of the tissue portion 10 facing the perfusion flow path 13 through the through hole 42d of the connector 42B, and as an example. The lumen 15 is formed by culturing for a certain period of time in a CO ₂ atmosphere at 37 ° C. and 5%. When culturing the vascular cells 14, the end of the pipe 63 downstream of the filter 62 shown in FIG. 1 is connected to the medium supply unit 66 in the pipe 64 arranged outside the culture tank 41. Further, for the pipe 64 on the culture medium discharge side, the end of the pipe 63 on the side introduced into the pump 60 is connected.

Then, by driving the pump 60 shown in FIG. 1, the medium M stored in the medium reservoir 61 is supplied to the perfusion flow path 13 after passing through the filter 62. Further, the medium M discharged from the perfusion channel 13 is returned to the pipe 63, foreign matter and the like are removed by the filter 62, and then supplied to the perfusion channel 13 for circulation. The medium M used is replaced every 2-3 days until the lumen 15 is concentrated, for example, 70-90%. As a result, the perfusion flow path 13 is surrounded by the lumen portion 15, and an artificial three-dimensional tissue 1 in which the tissue portion 10 is arranged outside the lumen portion 15 is formed.

(Barrier function measurement)
Next, a method of measuring the barrier function of the formed artificial three-dimensional tissue 1 will be described with reference to FIG.

As described above, the first tissue electrode 81 in the tissue electrode 80 is arranged in the tissue portion 10 in an exposed state, and the first flow path electrode 71 in the flow path electrode 70 is exposed in the perfusion flow path 13 and the medium M. It is arranged in the state of being. The measuring unit 90 measures the current value via the first tissue electrode 81 and the first flow path electrode 71 in a state where the medium M is perfused into the perfusion flow path 13.

Further, the second tissue electrode 82 in the tissue electrode 80 has an exposed end surface arranged in the through hole 42d and comes into contact with the tissue portion 10, and the second flow path electrode 72 in the flow path electrode 70 has an exposed end surface in the through hole 42d. It is placed in contact with the medium M. The measuring unit 90 measures the voltage value via the second tissue electrode 82 and the second flow path electrode 72 in a state where the medium M is perfused into the perfusion flow path 13. The internal impedance in the measuring unit 90 has a sufficiently large internal impedance as compared with the electrical resistance (transendothelial resistance) of the lumen portion 15.

Then, the measuring unit 90 uses the current value measured through the first tissue electrode 81 and the first flow path electrode 71 and the voltage value measured via the second tissue electrode 82 and the second flow path electrode 72. The electrical resistance value (transendothelium resistance value) between the tissue portion 10 and the medium M in the perfusion channel 13 is measured by the four-terminal method.

For example, when the electric resistance value is measured by the two-terminal method via the first tissue electrode 81 and the first flow path electrode 71, the voltage drop due to the resistance of the electrode itself and the contact resistance with the tissue portion 10 and the medium M is an error. The electrical resistance value included as is to be measured. On the other hand, since the internal impedance in the measuring unit 90 is large, almost no current flows through the second tissue electrode 82 and the second flow path electrode 72. Therefore, the voltage value can be measured with high accuracy without being affected by the voltage drop due to the resistance of the electrode itself and the contact resistance with the tissue portion 10 and the medium M.

FIG. 13 shows the case where the vascular cells 14 are injected (seed) in the artificial three-dimensional tissue 1 and the culture medium is perfused and cultured after about one day (perfused), and the culture medium is not perfused. It is a figure which shows the relationship between the culture time and trans-endothelial electrical resistance (TEER) in the case of culturing in (non-perfused).

As shown in FIG. 13, after seeding the vasculature cells 14, transendothelium electrical resistance increased until the perfusion of the medium was started. It is considered that this is because the vasculature cells 14 increased and covered the surface of the tissue portion 10 facing the perfusion channel 13, which is consistent with the observation result.

Then, one day after sowing, there was a difference in transendothelial electrical resistance depending on the presence or absence of perfusion of the medium. It is considered that this was caused by the difference in the growth state of the vasculature cells 14 due to the difference between the nutrient supply by perfusion of the medium and the cutoff of the nutrient supply by non-perfusion during culturing.
That is, in the present embodiment, it was confirmed that the difference in transendothelial electrical resistance caused by the difference in the growth state of the vasculature cells 14 in the lumen 15 can be measured.

(Evaluation methods)
As yet another aspect, the present invention relates to a method for evaluating the irritation of a drug to a tissue portion 10 using the artificial three-dimensional tissue 1 of the present invention. The drug in the present invention includes drugs such as pharmaceuticals, cosmetics, quasi-drugs and the like. According to the evaluation method of the present invention, for example, the drug can be evaluated in an environment closer to the actual tissue portion 10 as compared with the conventional method. Further, the evaluation method of the present invention is extremely useful in, for example, dynamic evaluation of drugs having various molecular weights in the creation (screening) of new drugs, and evaluation in the development of cosmetics, quasi-drugs, and the like.

The evaluation method of the present invention can be carried out, for example, by contacting the drug with the artificial three-dimensional tissue 1 and measuring the response to the stimulus caused by the contact of the drug. The response can be measured, for example, by measuring the transdermal electrical resistance. The drug refers to a substance to be evaluated, and examples thereof include inorganic compounds and organic compounds.

Further, by using the barrier function measuring system 100 and the barrier function measuring method described above, when measuring the amount of the drug permeating into the tissue portion 10 when flowing into the lumen portion (blood vessel) 15, an artificial three-dimensional structure is used. The transendothelial electrical resistance in tissue 1 can be evaluated with respect to the transendothelial electrical resistance in a living body. Alternatively, by using the barrier function measuring system 100 and the barrier function measuring method described above, fluctuations in the barrier function are caused when a drug that affects the barrier function of the lumen (blood vessel) 15 is flowed into the lumen 15. Can be measured.
Further, by using the above-mentioned barrier function measuring method, it is possible to validate whether or not the barrier function is sufficiently constructed in the lumen portion of the barrier function measuring system 100.

As described above, in the present embodiment, the transendothelium resistance value and the index of the barrier function are measured using the artificial three-dimensional tissue 1 in which the tissue portion 10 is arranged outside the tubular cavity portion 15. , Suppresses the reliability of the obtained transendothelium resistance value due to non-uniform flow of the medium, as in the case of measuring the electrical resistance value using a two-dimensionally arranged tissue. can. Therefore, in the barrier function measuring system 100 and the measuring method of the present embodiment, highly reliable transendothelial electrical resistance can be obtained, and the barrier function can be evaluated with high reliability.

Further, in the present embodiment, four electrodes of the first flow path electrode 71, the second flow path electrode 72, the first tissue electrode 81, and the second tissue electrode 82 are used when measuring the transendothelium resistance value. Since the terminal measurement is performed, the influence of the resistance of the electrode itself and the contact resistance with the tissue part 10 and the medium M can be suppressed, and the trans-endothelial resistance value is measured and the barrier function is evaluated with higher accuracy and reliability. Is possible.

Further, in the present embodiment, the first flow path electrode 71 and the second flow path electrode 72 constituting the flow path electrode 70 form the first stranded wire 73, and the first structure electrode 81 and the first structure electrode 80 constituting the structure electrode 80 are formed. Since the two structure electrodes 82 form the second stranded wire 83, the handleability of the flow path electrode 70 and the handleability of the tissue electrode 80 are improved, and the workability when indexing the barrier function is improved. Further, since the outer peripheral surface of the second flow path electrode 72 forming the first stranded wire 73 and the outer peripheral surface of the second structure electrode 82 forming the second stranded wire 83 are coated with insulation, the first stranded wire 73 is provided. It is possible to prevent the electrodes from being short-circuited at each of the second stranded wire 83 and the second stranded wire 83.

In addition, in the present embodiment, since the flow path electrode 70 is connected to one end side of the flow path forming member 43, the flow path electrode 70 can be easily removed by removing the flow path forming member 43 to the other end side. It can be arranged in the perfusion channel 13. Further, in the present embodiment, the pipe 64 mounted on the connector 42B has an insertion hole 65a through which the flow path electrode 70 is inserted and a supply hole 66a into which the medium is supplied, so that the pipe 64 is connected to the measurement unit 90. It is possible to easily supply the medium to the perfusion flow path 13 in which the flow path electrode 70 in the laid state is arranged.

Further, in the present embodiment, the extracellular matrix component 11 engages with the engaging portion 42c having a surface facing the side wall 44 provided with the connectors 42A and 42B, thereby suppressing the contraction of the extracellular matrix component 11. Since the tissue portion 10 is formed, even when the extracellular matrix component 11 has a large contraction, it is possible to stably produce a high-quality artificial three-dimensional tissue 1 by retaining the perfusion flow path 13.

Although the preferred embodiments of the present invention have been described in detail above, the present invention is not limited to such specific embodiments, but is within the scope of the gist of the present invention described within the scope of the claims. Various modifications and changes are possible.

For example, in the above embodiment, a configuration in which vascular cells 14 are cultured to form a lumen (blood vessel) 15 is exemplified, but the configuration is not limited to this configuration. For example, cells of a luminal organ system such as the esophagus and the intestine may be cultured to form the luminal portion 15.

Further, in the above embodiment, the configuration in which the extracellular matrix component 11 is cultured to form the tissue portion 10 is exemplified, but the configuration is not limited to this configuration. For example, extracellular matrix component 11 including nerve cells, cerebral vascular endothelial cells, astrocytes, and vascular pericytes may be cultured to form a tissue portion. In this case, the blood-brain barrier permeability can be evaluated by measuring the transendothelial electrical resistance using the barrier function measuring system 100 described above.
Since it is difficult to collect cells from the human brain, the cells required for constructing the blood-brain barrier model may be cells differentiated from iPS cells. Examples of cells used for reprogramming into iPS cells include fat cells, chondrocytes, osteoblasts, blood cells, fibroblasts and the like.

Further, in addition to the nervous system cells, it may be a muscle tissue using fibroblasts, skeletal muscle cells or cardiomyocytes. Further, it may be a liver tissue using hepatocytes or a pancreatic tissue using pancreatic cells. When the present invention is applied to such a tissue, the extracellular matrix used in the above embodiment does not necessarily have to exist, and may be one in which only various cells are accumulated.

Further, in the above embodiment, the configuration in which the transendocardial electrical resistance is measured by 4-terminal measurement is exemplified, but the configuration is not limited to this configuration, and the trans-endothelial electrical resistance may be measured by 2-terminal measurement. When performing two-terminal measurement, the resistance of the electrode itself and the contact resistance with the tissue part 10 and the medium M are used as correction values in advance by experiments or simulations, and the measured transendothelial electrical resistance is the above correction value. It should be corrected with.

Further, in the above embodiment, the first flow path electrode 71 and the second flow path electrode 72 form the first stranded wire 73, and the first tissue electrode 81 and the second structure electrode 82 constituting the tissue electrode 80 are the second. Although the configuration for forming the stranded wire 83 is illustrated, the configuration is not limited to this configuration, and the configuration may be arranged so as to be spaced apart from each other.

Further, the positions where the second flow path electrode 72 and the second tissue electrode 82, which are the voltage reference electrodes shown in the above embodiment, are arranged, that is, the exposed end faces of the second flow path electrode 72 and the second tissue electrode 82. Is placed as an example, the measurement position of the second flow path electrode 72 may be any position of the perfusion flow path 13, and the measurement position of the second tissue electrode 82 may be any position of the tissue portion 10. There may be.

Further, in the above embodiment, the flow path electrode 70 is connected to one end of the flow path forming member 43, and the flow path electrode 70 is pulled into the perfusion flow path 13 by moving the flow path forming member 43 to the other end side. However, the present invention is not limited to this configuration. For example, the flow path electrode 70 may be inserted into the internal space of the tubular flow path forming member 43, and the flow path electrode 70 may be exposed to the perfusion flow path 13 when the flow path forming member 43 is removed. ..

Further, in the above embodiment, the configuration in which the lumen portion 15 is formed linearly is exemplified, but the configuration may be such that the lumen portion 15 is formed in a Y-shape that branches in the middle. In this case, the flow path forming member 43 is composed of a base portion orthogonal to one of the side wall 44 and two branch portions that branch from one end of the base portion and extend to the other side of the side wall 44 and are separable from the base portion. The Y-shaped perfusion channel 13 can be formed by moving the base portion to one side of the side wall 44 and the branch portion to the other side of the side wall 44. When this configuration is adopted, the base portion and the branch portion may be formed in a cylindrical shape as described above, and the flow path electrode 70 may be inserted into the internal space of the base portion and the branch portion.

According to the present invention, a barrier function measuring system for an artificial three-dimensional tissue, a barrier function measuring method for an artificial three-dimensional tissue, and an artificial three-dimensional tissue capable of measuring transendothelial electrical resistance and evaluating a barrier function with high reliability are provided. The drug evaluation method used can be provided. Therefore, the present invention is useful in the fields of cosmetics, pharmaceuticals, pharmaceuticals and the like, for example.

1 ... Artificial three-dimensional tissue, 10 ... Tissue part, 11 ... Extracellular matrix component, 13 ... Perfusion channel, 14 ... Vascular cells, 15 ... Cavity part, 40 ... Perfusion device (device), 41 ... Culture tank, 42 ... Connector (support part), 42A ... Connector (first support part), 42B connector (second support part), 42c ... Engagement part, 42d ... Through hole (insertion hole), 43 ... Flow path forming member, 44 ... Side wall, 45 ... culture space, 60 ... pump (supply device), 70 ... flow path electrode, 71 ... first flow path electrode, 72 ... second flow path electrode, 80 ... tissue electrode, 81 ... first tissue electrode, 82 ... 2nd tissue electrode, 90 ... Measuring unit, 100 ... Barrier function measuring system, M ... Medium

Claims (20)

A lumen with a perfusion channel extending in a predetermined direction,
Tissues located outside the lumen and
A supply device that supplies the medium to the perfusion channel and
The flow path electrodes arranged in the perfusion flow path and
The tissue electrodes arranged in the tissue portion and
A measuring device for measuring transendothelial electrical resistance via the flow path electrode and the tissue electrode,
A barrier function measurement system for artificial three-dimensional tissues characterized by being equipped with. The flow path electrode has a first flow path electrode and a second flow path electrode arranged in the perfusion flow path.
The tissue electrode has a first tissue electrode and a second tissue electrode arranged in the tissue portion.
The measuring device is based on a current value measured through the first flow path electrode and the first tissue electrode, and a voltage value measured through the second flow path electrode and the second tissue electrode. The barrier function measuring system for an artificial three-dimensional tissue according to claim 1, wherein the transdermal electrical resistance is measured. The first flow path electrode and the second flow path electrode whose outer peripheral surface is insulated and coated form a first stranded wire twisted to each other.
The barrier function measurement of the artificial three-dimensional structure according to claim 2, wherein the first structure electrode and the second structure electrode whose outer peripheral surface is insulated and coated form a second stranded wire twisted to each other. system. A device for culturing the artificial three-dimensional tissue is provided.
The device includes a culture tank having a culture space surrounded by a side wall and a culture tank.
A first support portion that supports the tissue electrode suspended along the predetermined direction in a region in the culture space where the artificial three-dimensional tissue is arranged through the opposite side wall.
A second support portion for attaching and detachably supporting an axial flow path forming member suspended along a predetermined direction in a region in the culture space where the artificial three-dimensional tissue is arranged through the opposite side wall. Have,
Any one of claims 1 to 3, wherein the perfusion flow path is formed in a space from which the flow path forming member is removed from the artificial three-dimensional structure, and the flow path electrode is arranged. Barrier function measurement system for artificial three-dimensional tissue as described in the section. One end of the flow path electrode is connected to one end of the flow path forming member, and the flow path electrode is connected to one end.
The barrier function measuring system for an artificial three-dimensional tissue according to claim 4, wherein the flow path electrode is arranged in the perfusion flow path when the flow path forming member is removed to the other end side. The claim is characterized in that the first support portion and the second support portion each have an engaging portion that engages with the artificial three-dimensional tissue and suppresses the contraction of the artificial three-dimensional structure in the predetermined direction. The barrier function measuring system for artificial three-dimensional tissue according to 4 or 5. The second support portion has an insertion hole through which the flow path forming member and the flow path electrode are inserted.
The barrier function measuring system for an artificial three-dimensional tissue according to any one of claims 4 to 6, wherein the supply device supplies a medium to the perfusion channel through the insertion hole. The barrier function measuring system for an artificial three-dimensional tissue according to any one of claims 1 to 7, wherein the lumen is formed using vascular cells. The barrier function measuring system for an artificial three-dimensional tissue according to any one of claims 1 to 8, wherein the tissue portion contains an extracellular matrix component. The barrier function measuring system for an artificial three-dimensional tissue according to claim 9, wherein the tissue portion contains nervous system cells. To form an artificial three-dimensional tissue having a lumen having a perfusion flow path extending in a predetermined direction and a tissue having a tissue located outside the lumen.
Supplying the medium to the perfusion channel and
A method for measuring a barrier function of an artificial three-dimensional tissue, which comprises measuring transendothelial electrical resistance via a flow path electrode arranged in the perfusion flow path and a tissue electrode arranged in the tissue portion. The flow path electrode has a first flow path electrode and a second flow path electrode arranged in the perfusion flow path.
The tissue electrode has a first tissue electrode and a second tissue electrode arranged in the tissue portion.
The measurement of the transendothelial electrical resistance is measured through the first flow path electrode and the first tissue electrode, and the current value measured through the second flow path electrode and the second tissue electrode. The method for measuring the barrier function of an artificial three-dimensional structure according to claim 11, wherein the transdermal electrical resistance is measured based on the voltage value. The first flow path electrode and the second flow path electrode whose outer peripheral surface is insulated and coated form a first stranded wire twisted to each other.
The barrier function measurement of the artificial three-dimensional structure according to claim 12, wherein the first structure electrode and the second structure electrode whose outer peripheral surface is insulated and coated form a second stranded wire twisted to each other. Method. A device including a culture tank having a culture space surrounded by a side wall, an axial flow path forming member suspended along the predetermined direction in the culture space through the opposite side wall, and the tissue electrode. To prepare and
By culturing cells in the culture space to form the tissue portion through which the flow path forming member and the tissue electrode penetrate.
To form the perfusion flow path penetrating the artificial three-dimensional tissue by removing the flow path forming member from the artificial three-dimensional tissue.
By arranging the flow path electrode in the perfusion flow path,
The artificiality according to any one of claims 11 to 13, wherein vascular cells are seeded on the surface of the tissue portion facing the perfusion flow path to form the lumen portion, and the present invention comprises. A method for measuring the barrier function of a three-dimensional tissue. The device has a first support portion that supports the tissue electrode suspended along the predetermined direction, and a second support that attaches and detachably supports the flow path forming member suspended along the predetermined direction. Has a part and
A part of the artificial three-dimensional tissue is engaged with the engaging portions provided on the first support portion and the second support portion, respectively, to form the tissue portion while suppressing the contraction in the predetermined direction. The method for measuring the barrier function of an artificial three-dimensional tissue according to claim 14. The second support portion has an insertion hole through which the flow path forming member and the flow path electrode are inserted.
The method for measuring the barrier function of an artificial three-dimensional tissue according to claim 15, wherein supplying the medium to the perfusion channel is to supply the medium to the perfusion channel through the insertion hole. By arranging the flow path electrode in the perfusion flow path, the flow path forming member having one end of the flow path electrode connected to one end thereof is removed to the other end side, whereby the flow path electrode is perfused. The method for measuring a barrier function of an artificial three-dimensional tissue according to any one of claims 14 to 16, wherein the method comprises arranging the artificial three-dimensional tissue on a road. The method for measuring a barrier function of an artificial three-dimensional tissue according to any one of claims 11 to 17, wherein the tissue portion contains an extracellular matrix component. The method for measuring a barrier function of an artificial three-dimensional tissue according to any one of claims 11 to 18, wherein the tissue portion contains nervous system cells. To measure the barrier function of the artificial three-dimensional tissue by the measuring method according to any one of claims 11 to 19.
Contacting the drug with the artificial three-dimensional tissue and
To measure the response of the artificial three-dimensional tissue to the stimulus by the contact of the drug,
A drug evaluation method using an artificial three-dimensional tissue characterized by containing.

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